Abstract
Unlike plants and invertebrates, mammals reportedly lack proteins displaying asparagine (N)-linked paucimannosylation (mannose(1-3)fucose(0-1)N-acetylglucosamine(2)Asn). Enabled by technology advancements in system-wide biomolecular characterization, we document that protein paucimannosylation is a significant host-derived molecular signature of neutrophil-rich sputum from pathogen-infected human lungs and is negligible in pathogen-free sputum. Five types of paucimannosidic N-glycans were carried by compartment-specific and inflammation-associated proteins of the azurophilic granules of human neutrophils including myeloperoxidase (MPO), azurocidin, and neutrophil elastase. The timely expressed human azurophilic granule-resident β-hexosaminidase A displayed the capacity to generate paucimannosidic N-glycans by trimming hybrid/complex type N-glycan intermediates with relative broad substrate specificity. Paucimannosidic N-glycoepitopes showed significant co-localization with β-hexosaminidase A and the azurophilic marker MPO in human neutrophils using immunocytochemistry. Furthermore, promyelocyte stage-specific expression of genes coding for paucimannosidic proteins and biosynthetic enzymes indicated a novel spatio-temporal biosynthetic route in early neutrophil maturation. The absence of bacterial exoglycosidase activities and paucimannosidic N-glycans excluded exogenous origins of paucimannosylation. Paucimannosidic proteins from isolated and sputum neutrophils were preferentially secreted upon inoculation with virulent Pseudomonas aeruginosa. Finally, paucimannosidic proteins displayed affinities to mannose-binding lectin, suggesting immune-related functions of paucimannosylation in activated human neutrophils. In conclusion, we are the first to document that human neutrophils produce, store and, upon activation, selectively secrete bioactive paucimannosidic proteins into sputum of lungs undergoing pathogen-based inflammation.
Highlights
Protein paucimannosylation is considered an important invertebrate- and plant-specific glycoepitope
In line with their presence in specific micro-environments that are central to inflammation and pathogen infection, we confirm that the timely expressed human azurophilic granule-resident -hexosaminidase A (Hex A) enzymatically facilitates the generation of protein paucimannosylation by trimming hybrid/complex type N-glycan intermediates using a machinery, which is formed during early myeloid maturation, and functionally associate paucimannosidic proteins with roles in innate immunity upon secretion from activated human neutrophils
Mucoid/non-mucoid P. aeruginosa, A. fumigatus, S. pneumoniae, and S. aureus were identified in the pathogen-infected paucimannose-rich sputum (Table 1), illustrating a pathogen species-unspecific link to paucimannosylation
Summary
Protein paucimannosylation is considered an important invertebrate- and plant-specific glycoepitope. Enabled by recent developments in system-wide biomolecular detection, we document that inflammation-associated proteins, localizing to the azurophilic granules of human neutrophils, abundantly display paucimannosylation In line with their presence in specific micro-environments that are central to inflammation and pathogen infection, we confirm that the timely expressed human azurophilic granule-resident -hexosaminidase A (Hex A) enzymatically facilitates the generation of protein paucimannosylation by trimming hybrid/complex type N-glycan intermediates using a machinery, which is formed during early myeloid maturation, and functionally associate paucimannosidic proteins with roles in innate immunity upon secretion from activated human neutrophils
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